Echinops taeckholmiana Amin: Optimization of a Tissue Culture Protocol, Biological Evaluation, and Chemical Profiling Using GC and LC-MS.

There have been no reports published on the rare Echinops taeckholmiana growing wildly in Egypt. So, this study aimed to preserve it through method optimization of in vitro seed germination, besides callus formation from induced seedlings. Chemical profiling using gas chromatography-mass spectrometry (GC-MS) analysis of the n-hexane fraction afforded 29 identified components, mainly fatty acids esters, sesquiterpenes, triterpenes, one thiophene, and bis(2-ethylhexyl) phthalate. Ultra-performance liquid chromatography-electron spray ionization/tandem mass spectrometry (UPLC-ESI/MS-MS) analysis of total alcoholic root and induced calli extracts resulted in 68 metabolites. Taraxeryl acetate, β-sitosterol, stigmasterol-3β-d-glucoside, and 1,1,1-kestopentaose were identified from the defatted root extract, which inhibited α-amylase (IC50 54.6 μg/mL) and α-glucosidase (60.4 μg/mL) enzymes compared with acarbose (IC50 values of 30.57 and 34.71 μg/mL, respectively). Moreover, it showed moderate activity against HepG2 (IC50 31.1 ± 1.4 μg/mL) and no activity against M-NFS-60 cell lines compared to cisplatin (IC50 3.25 ± 1.4 and 8.37 ± 0.25, respectively).

Introduction

Genus Echinops L. family Asteraceae is the target of the present study (tribe Echinopeae Cass) and can be easily distinguished by its spinescent leaves and compound capitula built up of numerous uniflorous heads.[1] Täckholm, distinguished between five Egyptian Echinops species, namely, Echinops glaberrimus DC., Echinops hussoni Boiss., Echinops macrochaetus Fresen., Echinops spinosissimus Turra (= Echinops spinosus L.,) and Echinops galalensis Schweinf.[2] Later, the Egyptian Echinops taeckholmiana Amin, which is endemic to the Northwest Nile Delta, was described.[3]

E. taeckholmiana is a spiny perennial decumbent whitish green herb up to 40 cm in height with white tomentose, especially in the upper parts. The leaves are pinnatisect; lobes are narrowly linear with revolute, spiny margins measuring 3.5–8 cm. Synflorescences measured about 1.7–2.5 cm in diameter with whitish cream corolla. Phyllaries formed of about 12 bracts arranged in three whorls and measured about 5–6 mm, 1 cm, and 5 mm for the outer, middle, and inner bracts, respectively (Figure ).[4]

Figure 1

E. taeckholmiana: (A) habit; (B) capitula; (C) floret; (D) bracts; (E) outer, median, and inner phyllaries. “Illustration courtesy of “Magdy El-Gohary.” Copyright 2021.”.

Preceding chemical investigation on the genus Echinops revealed the presence of thiophenes, quinoline alkaloids, sesquiterpenes (hydrocarbons and lactones), triterpenes, flavonoids, lignans, and volatile oils resulted in their hepatoprotective, anti-inflammatory, antifungal, antifeedant, nematocidal, and cytotoxic activities.[5]

The in vitro propagation and callus induction of wild medicinal plants have become important tools to preserve endangered species and produce active metabolites in sufficient amount and quality.[6]

The up-to-date survey proved that there have been no reports published on phytochemical and biological investigation, in vitro seed germination, and callus induction of E. taeckholmiana Amin. Therefore, the current study aims to study the ability of the plant seeds to germinate in vitro and the ability of the seedlings to induce callus. Besides, profiling of the total defatted root and induced calli extracts was performed using ultra-performance liquid chromatography-electron spray ionization/tandem mass spectrometry (UPLC-ESI-MS/MS). Additionally, gas chromatography-mass spectrometry (GC-MS) and column chromatography as well as cytotoxic and in vitro antidiabetic activities were investigated.

Results and Discussion

In Vitro Seed Germination

Sterilization of the seeds using 70% ethyl alcohol (2 min) and a 5% hypochlorite solution (5 min) afforded a high-seed decontamination rate (98%) with a seed germination percentage of 75% (Figure ).

Figure 2

Germination of E. taeckholmiana seeds over hormone-free, half-strength, MS media: (a) 10 days old seedling; (b) 20 days old seedling; and (c) 30 days old seeding.

Callus Induction and Maintenance

Medium I (MS media supplied with 1 mg/L 2,4D and 0.5 mg/L kinetin) exhibited the shortest callus initiation time (9 days) and 100% callus induction percentage (Table and Figure ). Moreover, medium I showed the best callus growth rate compared to other media. The growth curve of medium I (Figure ) showed a short lag phase during which a small increase in the fresh weight was observed. After that, it showed an exponential phase through which the fastest growth rates of the calli were observed. Also, medium I showed the best growth parameters with GI = 1.38, μ = 0.023, and doubling time (dt) = 30.75 days (Table and Figure ). On the other hand, media II and III showed weak growth with low growth indices and long doubling time.

Table 1

Callus Initiation and Callus Induction Percentages for Different Media Compositions

media no.	media composition	callus initiation days	callus induction %
medium I	MS + 1 mg/L 2,4D and 0.5 mg/L kinetin	9	100
medium II	MS + 1 mg/L NAA and 0.1 mg/L BAP	9	100
medium III	MS + 1 mg/L NAA and 0.5 TDZ	12	90

Figure 3

Seven weeks old callus of E. taeckholmiana grown on MS media supplemented with different phytohormones: (A) medium I; (B) medium II; and (C) medium III.

Figure 4

Growth curves of calli during 6 weeks grown on MS media supplemented with different phytohormones.

Table 2

Callus Fresh Weight (g) and Different Growth Parameters of Callus Grown on MS Media Supplemented with Different Phytohormones

	callus fresh weight (g)
media no.	7 days	14 days	21 days	28 days	35 days	42 days	growth index (GI)	specific growth rate (μ) g/day	doubling time (dt)
medium I	2.19	1.38	0.023	3.76	4.53	4.76	1.38	0.023	30.75
medium II	2.19	0.56	0.014	2.91	3.24	3.30	0.56	0.014	49.23
medium III	2.03	0.49	0.010	2.73	2.83	2.98	0.49	0.010	71.44

Characterization of the Isolated Compounds

Four compounds (Supporting Text ST1–4 and Figure ) were isolated from the defatted root extract and identified as taraxeryl acetate (1),[7]β-sitosterol (2),[8] stigmasterol-3-β-d-glucoside (3),[9,10] and O-β-d-fructofuranosyl-(2-1)-β-d-fructofuranosyl-(2-1)-β-d-fructofuranosyl-(2-1)-β-d-fructofuranosyl-α-d-glucopyranose, commonly known as 1,1,1-kestopentaose (4),[11] based on their physical characteristics and spectral data (mass and 1H and 13C NMR; Figures 1S–10S).

Figure 5

Structures of isolated compounds from the root extract of E. taeckholmiana.

Characterization of Gas–Liquid Chromatography-Mass Spectrometry

The GC-MS chromatogram (Figure 11S) of the n-hexane fraction afforded the identification of 29 compounds (Table ), representing 95.26%. Ethyl and methyl esters of fatty acids are considered the major constituents (46.86%) including ethyl palmitate (19.30%), ethyl linoleate (14.24%), and methyl palmitate (8.30%). Additionally, sesquiterpenes such as vatirenene (0.54%) and jatamol A (0.48%) and triterpenes (taraxerone, 0.17% and traxeryl acetate, 0.45) were identified. Furthermore, 2,2′,5′,2″-terthiophene (4.65%) is common in Echinops species and known for its antifungal and cytotoxic activities.[12,13] Finally, bis(2-ethylhexyl) phthalate (a plasticizer and an environmental pollutant) was detected in a considerable amount (12.42%), and found to be synthesized inside plants and microorganisms.[14,15]

Table 3

Compounds Identified in the n-Hexane Soluble Fraction of the E. taeckholmiana Root Using GC-MS

peak no.	Rt (min)	relative area %	mol formula	[M+] m/z	component	ref
1	18.2	0.16	C14H28	196	1-tetradecene	(16)
2	19.3	0.23	C11H22O2	185	decanoic acid methyl ester (methyl decanoate)	(17)
3	20.2	0.25	C14H20O2	220	2,5-cyclohexadiene-1,4-dione-2,6-bis(1,1-dimethylethyl)	(18)
4	21.33	4.02	C14H22O	206	phenol, 2,4-bis(1,1-dimethylethyl)
6	23.2	1.97	C14H22O	206	phenol, 3,5-bis(1,1-dimethylethyl)
7	24.1	1.05	C13H10O	182	benzophenone	(19)
11	27.6	1.42	C18H36	252	1-octadecene	(20)
12	28.5	0.32	C16H32O2	256	pentadecanoic acid methyl ester (methyl pentadecanoate)
13	28.9	0.54	C15H22	202	vatirenene sesquiterpene
14	29.3	0.48	C15H24O	220	β-selinen-2α-ol (jatamol A)
15	29.6	0.78	C17H34O2	270	pentadecanoic acid ethyl ester (ethyl pentadecanoate)
16	30.4	8.3	C17H34O2	270	hexadecanoic acid methyl ester (methyl palmitate)	(21)
17	31.97	19.3	C18H36O2	284	hexadecanoic acid ethyl ester (ethyl palmitate)	(22)
18	33.7	7.11	C18H32O2	280	linoleic acid	(23)
19	35.1	14.24	C20H36O2	308	linoleic acid ethyl ester (ethyl linoleate)
20	35.9	4.65	C12H8S3	248	2,2′,5′,2″-terthiophene
22	37.5	0.53	C21H42O2	326	arachidic acid methyl ester (eicosanoic acid methyl ester)	(24)
23	38.7	1.55	C22H44O2	340	arachidic acid ethyl ester (eicosanoic acid ethyl ester)
24	41.4	12.42	C24H38O4	390	bis(2-ethylhexyl) phthalate	(25)
25	42	4.29	C24H26O	330	phenol, 2,4-bis(1-methyl-1-phenylethyl)
26	43.7	0.44	C25H50O2	382	tetracosanoic acid methyl ester (methyl lignocerate)	(26)
27	44.6	1.17	C26H52O2	396	ethyl lignocerate
28	51.4	0.17	C30H48O	424	taraxerone	(27)
29	53.4	0.45	C32H52O2	468	traxer-14-en-3-β-yl acetate

Characterization of UPLC-ESI-MS/MS

Profiling of alcoholic root and callus extracts was carried out by UPLC-ESI-MS/MS (Figure 12S). The compounds were identified based on the retention time (Rt), mass, MS2, comparison with the standards, reported literature, and database (TMIC and MassBank). In total, 68 compounds were tentatively identified (phenolic acids and their derivatives, carbohydrates, flavonoids, sterols, and terpenes), 48 in the negative mode and 51 in the positive mode, of which 13 could be detected in both modes. All identified compounds were ordered according to relative retention times to 3,5-dicaffeoylquinic acid and are listed in Table .

Table 4

Metabolites Identified in the Root and Callus of E. taeckholmiana Using UPLC-ESI-MS/ MS

no	RRt. (min)	m/z [M – H] –/[M + H]+	major fragments (m/z)	compound name	ERa	ECb
1	0.097	191/-----	173	quinic acid	+	+
2	0.099	133/-----	115, 71	malic acid	+	+
3	0.101	515/517	353, 191, 179	3,5-dicaffeoylquinic acid	+	–
4	0.106	195/-----	177, 159, 129(100%)	gluconic acid	+	+
5	0.111	377/-----	333, 283, 271, 257, 187, 163, 119	coumaroylquinic acid	+	+
6	0.191	163/165	119	p-coumaric acid	+	+
7	0.200	353/355	707 [2M – H]−, 191, 178.9	3-caffeoylquinic acid	+	+
8	0.257	349/-----	173	feruloylshikimic acid	+	+
9	0.264	178.9/-----	161, 119, 89	hexose sugar	+	+
10	0.278	179/-----	161, 135	caffeic acid	+	–
11	0.575	-----/389	226, 182	dihydrosinapoyl-O-glucoside	–	+
12	0.847	193/-----	178, 149, 134	ferulic acid	+	–
13	1	515/517	353, 335, 179	3,4-dicaffeoylquinic acid	+	+
14	1.248	163/165	119	O-coumaric acid	+	+
15	1.252	515/517	353, 299	4,5-dicaffeoylquinic acid	+	+
16	1.368	-----/611	609, 477, 315	quercetin methyl ether pentoside hexoside	+	–
17	1.370	-----/433	385	1-O-d-glucopyranosyl sinapate	–	+
18	1.370	609/611	609, 301	rutin	+	–
19	1.406	207/209	179, 159, 135, 127 103	ethyl caffeate	+	–
20	1.417	529/-----	529, 501, 367, 193	feruloyl caffeoylquinic acid derivative	–	+
21	1.421	499/-----	337, 193	coumaroyl caffeoylquinic acid	–	+
22	1.427	311/313	179, 149	caftaric acid	+	+
23	2.007	335/-----	178.8	O-caffeoylshikimic acid isomer	–	+
24	2.347	353/355	191	5-caffeoylquinic acid	+	–
25	2.351	367/369	191, 173 175	feruloylquinic acid derivative	–	+
26	2.472	293/295	236, 221, 193	ferulic acid derivatives	+	–
27	2.481	349/----	193	feruloylshikimic acid isomer	+	+
28	2.611	353/355	172	4-caffeoylquinic acid	+	–
29	2.658	371/----	371, 354(100%), 209	caffeoylglucaric acid	–	+
30	2.660	331/333	271, 169(100%)	1-galloyl-O-glucoside	+	+
31	2.823	----/679	515, 353	tricaffeoylquinic acid derivative	+	–
32	2.979	----/287	285, 257, 151, 169	kaepmpherol	+	+
33	3.108	----/305	302, 285, 125, 178	taxifolin	–	+
34	3.115	----/355	191, 172	caffeoylquinic acid derivative	–	+
35	3.123	----/317	315, 300, 271, 255, 179	quercetin-3-O-methyl ether	+	–
36	3.151	----/317	300(100%), 245	isorhamnetin	–	+
37	3.184	----/301	299, 284	trihydroxy methoxy flavone	+	–
38	3.198	335/----	190.8, 178.9, 135	3-O-caffeoylshikimic acid	+	+
39	3.274	----/829	667, 505, 343, 181	1,1,1-kestopentaosec	+	+
40	3.337	313/----	313, 298, 285	kaempferol dimethyl ether	+	+
41	3.446	----/317	317, 302, 195, 167	dihydroxy dimethoxy flavanone	+	+
42	3.533	315/----	153 [(M – H)- glu]	protocatechuic acid hexoside	+	+
43	3.557	441/443	289, 169, 135	(epi) Catechin gallate	+	–
44	3.571	----/319	299 [100%], 179, 151	methyldihydroquercetin	–	+
45	3.583	295/----	295[100%], 277, 195, 179,	monohydroxy-octadecadienoic acid (OH-18:2)	+	–
46	3.729	----/301	301, 285, 272	kaempferol-3-O-methyl ether	+	–
47	3.736	341/343	179	dihexoside	+	+
48	3.738	317/319	317, 179, 151	dimethyl (epi) catechin	+	+
49	3.953	----/345	191, 169, 125	3-O-galloylquinic acid	+	–
50	3.998	----/307	179, 165, 125	gallocatechin	+	–
51	4.049	337/----	191	p-coumaroylquinic acid	–	+
52	4.080	----/355	191	caffeoylquininc acid derivative	–	+
53	4.094	447/----	301	quercetin-3-O-rhamnoside	–	+
54	4.146	339/----	339, 177	esculetin-6-O-glucoside	+	+
55	4.156	----/301	272, 179, 151	quercetin	+	+
56	4.179	377/----	377, 317, 275, 257	myricetin monoacetate	+	–
57	4.269	----/355	179	caffeoylquinic acid derivative	+	+
58	4.321	515/----	353, 179	dicaffeoylquinic acid derivative	+	+
59	4.375	----/379	335, 273, 165,	coumaroylquinic acid isomer	+	–
60	4.512	255/----	257, 239, 229, 221, 213, 211, 209	palmitic acid	–	+
61	4.526	305/307	261, 219, 221	epigallocatechin	+	–
62	4.671	----/309	291, 237, 187	dihydroxy-octadecatetraenoic acid	+	–
63	4.788	----/287	285, 267, 213, 151, 133	luteolin	+	–
64	4.927	501/503	341, 179	inulin derivatives	+	+
65	4.988	665/667	503, 341, 179	inulin derivative	+	+
66	5.217	---- /413	412, 394, 379, 271	stigmasterol	+	+
67	5.226	---- /415	414, 396, 381, 273,	β-sitosterolc	+	+
68	5.354	---- /429	429, 387, 372	taraxeryl acetatec	+	–

Root extract.

Callus extract.

Compounds isolated from the root extract.

Carbohydrates and Sugars

Carbohydrates containing fructose with or without terminal glucose per molecule are known as fructans (synthesized through the addition reaction of fructose to sucrose). The trisaccharides obtained were 1-kestotriose (1-kestose), 6-kestotriose (6-kestose), and 6G-kestotriose (neokestose). Furthermore, inulin-type fructans (linear structure) were formed by further elongation of 1-kestose core with a β (2-1) fructose-fructosyl linkage that distributed the family Asteraceae as an energy source.[28] Hexose sugar (9), 1,1,1-kestopentaose (39), dihexoside (47), and inulin derivatives (64) and (65) were elucidated by comparing their pseudomolecular ions (179, 829, 343, 503, and 667, respectively) and MS2 with reported data.[29,30] Moreover, MS2 (667, 505, 343, and 181) of 1,1,1-kestopentaose exhibited successive loss of the fructose moiety [M-162].

Organic and Phenolic Acids and Their Derivatives

Malic acid (2, organic acid) and seven phenolic acids such as p-coumaric acid (6), ferulic acid (12), o-coumaric acid (14),[31] quinic acid (1),[32] gluconic acid (4),[33] caffeic acid (10), and caftaric acid (22)[34] were identified in the total root and callus extracts. Caftaric acid (22) showed [M – H]− at m/z 311 and MS2 at 179 [M – 132, tartaric acid residue]− and 149 [M – 162, caffeoyl moeity]−.

Additionally, esters of caffeic, ferulic, and coumaric acids with quinic and shikimic acids were identified. Compounds (3), (13), (15), and (58) showed [M – H]− at m/z 515 with distinctive MS2 at m/z 353 [M – H – caffeoyl] and 191 for the quinate moiety and were identified to be 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, and dicaffeoylquinic acid derivative, respectively.[35] Furthermore, mono-caffeolyquinic acid derivatives were suggested for compounds (7), (24), (28), (31), (34) (52), and (57) based on molecular ions at m/z 353/355. The published literature[35] demonstrated that compounds (7), (24), and (28) were identified as 3-caffeoylquinic acid, 5-caffeoylquinic acid, and 4-caffeoylquinic acid, respectively, while compounds (34, MS2, 191, 172), (52, MS2, 191 for quinates [M – 162]−), and (57, MS2, 179 [M – 174, caffeate]) were identified as caffeolyquinic acid derivatives. Compound (31) was identified as tricaffeoylquinic acid with [M + H] + at m/z 679 and MS2 at 515 [M + H – caffeoyl] + and 353 [M + H – 2caffeoyl] +.

Coumaroylquinic acid and its isomer were suggested for compounds (5, [M – H]− at m/z 377) and (59, [M + H] + at m/z 379), with distinctive MS2 at m/z 333, 283, 271, 187, 163, and 119 and 335, 273, and 165, for both compounds, respectively. Compound (51) was confirmed to be p-coumaroylquinic acid with [M – H]− at m/z 377 and a fragment ion at m/z 191 [M – 147, coumaroyl moiety]−.[36][36] Additionally, compound (21) was proposed to be coumaroyl caffeoylquinic acid (23), where it exhibited [M – H]− at m/z 499 with MS2 at m/z 377 [M – 162, caffeoyl moiety]− and 191 [M – 309, coumaryl caffeoyl moiety]−. Feruloylshikimic acid and its isomer were proposed for compounds (8) and (27), which were detected at m/z 349 [M – H]−, with MS2 at 173 [M – 193 – H2O]− for (8) and 193 for (27) (30, 36). Moreover, other three ferulic acid derivatives were detected at [M – H]− at m/z 529, 367, and 293, as well as MS2 at m/z 529, 501, 367, and 193 for compound (20); 191, 173, and 175 for compound (25); and 236, 221, and 193 for compound (26), hence elucidated as feruloyl caffeoylquinic acid, feruloylquinic acid, and ferulic acid derivatives, respectively.[30] Furthermore, esters of caffeoyl acid were detected at [M – H]− at m/z 235 for compounds (23) and (38) and 371 for compound (29), where compound (23) showed MS2 at m/z 179 (caffeic acid) and identified as the O-caffeoylshikimic acid isomer, while 3-O-caffeoylshikimic acid was elucidated for compound (38) based on MS2 at m/z 191, 179, and 135. However, compound (29) showed distinctive MS2 at m/z 354 [M – 17]−, 209, glucaric acid [M – 162, caffeoyl moiety]− and identified as caffeoylglucaric acid.[30] Also, compound (11) was identified as dihydrosinapoyl-O-glucoside from [M + H]+ at m/z 289 with MS2 at 226 and 182.[31] 1-O-d-glucopyranosyl sinapate was suggested for compound (17) showing [M + H]+ at 433, MS2 fragment ion at m/z 285, and reported data.[34] Compound (19) was detected at m/z 207/209 (both modes) with MS2 at 179, 159, 135, 127, and 103, hence identified as ethyl caffeate.[32] However, gallic acid derivative compounds (30), (43), (49), (50), and (61) were detected, where 1-galloyl-O-glucoside was suggested for (30) and (31) according to its molecular ion (m/z 331/ 333) and MS2 (271). (epi) Catechin gallate was proposed for (43) based on its molecular ion at m/z 441/443 and fragment ions at m/z 289 [M – galloyl moiety, 154]+, 169, and 135.[37] 3-O-Galloylquinic acid was proposed for compound (49) based on [M + H]+ at m/z 345 and MS2 at m/z 191 for quinic acid [M – galloyl moiety, 154]+, 169, and 125.[38] Finally, the detected [M + H]+ at m/z 307 for compounds (50) and (61) was identified as gallocatechin (MS2, 179, 165, and 125) and epigallocatechin (MS2, 261, 219, and 221), respectively.[39]

Protocatechuic acid hexoside (42)[33] and dimethyl (epi) catechin (48)[39] were confirmed based on [M – H]− (at m/z 315 and 317 with a peak at 635 [2M – H], respectively) and MS2 (153 [(M – H)– glu] and 179, 151, respectively).

Flavonoids and Their Glycosides

Quercetin methyl ether pentoside hexoside (16) exhibited [M + H] + at m/z 611 with MS2 at m/z 477 [M + H – pentoside]+ and 315 [M + H – hexoside]+.[40] Compound (18) was identified as rutin[39] based on molecular ions at m/z 609/611 and MS2 at m/z 301 [M + H – 308, rutinoside]+. Additionally, compound (32) detected at m/z 287 [M + H]+ and MS2 at 285, 257, 151, and 169 was suggested to be kaempferol.[40] Furthermore, compound (33) exhibited [M + H] + at m/z at 305 with MS2 at m/z 285, 125, and 178 and identified as taxifolin.[41] Compounds (35), (36), and (41) were recognized at similar [M + H] + at m/z 317 with distinctive fragments for quercetin-3-O-methyl ether (35) at m/z 315, 300, 271, and 255.[34] However, isorhamnetin (36) showed MS2 at m/z 300 (100%) and 245.[42] Moreover, dihydroxy dimethoxy flavanone (41) exhibited typically matched MS2 at m/z 317, 302, 195, and 167 with the literature.[40] [M + H]+ at m/z 301 was designated for three compounds (37, MS2, 299 and 284), (46, MS2, 285 and 272), and (55, MS2, 272, 179, and 151) that coincided with trihydroxy methoxy flavone,[31] kaempferol-3-O-methyl ether,[40] and quercetin,[32] respectively. Compound (40) that displayed [M – H]− at m/z 313 besides MS2 at m/z 298 and 285 (loss of two methyl) was characterized as kaempferol dimethyl ether.[40] Furthermore, based on [M – H]− at m/z 319 and MS2 at m/z 299 [M – H – 18], 179, and 151, compound (40) was confirmed as methyldihydroquercetin.[43] Quercetin-3-O-rhamnoside[44] was characterized for (53) based on [M – H]− at m/z 477 and MS2 (301, [M – 146]−, rhamnosyl). Moreover, esculetin-6-O-glucoside (54) was detected at m/z 339 [M – H]− with MS2 at m/z 177 [M – 162, glucose moiety]−.[45] Compound (56) showed [M – H]− at m/z 377 with characteristic fragments at m/z 317, 275, and 257 and was identified as myricetin monoacetate.[39] Finally, compound (63) was confirmed as luteolin, based on [M + H]+ at m/z 287 and MS2 at m/z 267, 213, and 151.[31]

Sterols and Terpenes

Compounds (66), (67), and (68) showed [M + H]+ at m/z 413, 415, and 429, respectively, with other additional fragments and identified as stigmasterol, β-sitosterol, and taraxeryl acetate, respectively, through the comparison of their MS2 with the reported data.[46,47]

Miscellaneous Compounds

Fatty acids were detected at m/z 295 [M – H]−, 255 [M – H]−, and 309 [M + H]+ and were confirmed to be monohydroxy-octadecadienoic acid (45),[48] palmitic acid (60),[49] and dihydroxy-octadecatetraenoic acid (62),[50] respectively.

In Vitroα-Amylase and α-Glucosidase Inhibitory Activities

Type II diabetes is a chronic disease that results in several metabolic disorders such as obesity, dyslipidemia, elevated blood pressure, atherosclerosis, and macro- and microvascular complications.[51] Drugs with α-amylase and α-glucosidase activities can retard the intestinal absorption of glucose to decrease postprandial blood sugar levels.[52]

The current antidiabetic activity of the root extract was evaluated (Table 1S and Figure ) and it exhibited α-amylase and α-glucosidase enzymes inhibitory activities (IC50 54.6 and 60.4 μg/mL) compared with acarbose (IC50, 30.57 and 34.71 μg/mL), respectively.

Figure 6

Percentage of inhibition of different concentrations of the alcoholic root extract and acarbose standard for α-amylase and α-glucosidase enzymes.

Cytotoxic Activity

The up-to-date survey proved that the cytotoxicity of the root extract against HepG2 and M-NFS-60 cell lines using the MTT cell viability assay was evaluated in this study for the first time. According to the protocols of the American National Cancer Institute (NCI),[53] the root extract exhibited moderate cytotoxic activity against the HepG2 cell line with an IC50 value of 31.1 ± 1.4 μg/mL compared with cisplatin as a reference standard (IC50 3.25 ± 1.4). However, the root extract has no activity against the M-NFS-60 cell line (IC50 121 ± 7.2 μg/mL) upon comparison with cisplatin (IC50 8.37 ± 0.25 μg/mL) (Figures and 8).

Figure 7

Cytotoxic effects of different concentrations of the E. taeckholmiana alcoholic root extract and cisplatin standard drug on cell viability of HepG2 and M-NFS-60 cell lines using the MTT assay (n = 3). Data were expressed as the mean value of cell viability (% of control) ± standard deviation (SD).

Figure 8

Cytotoxic effects of the E. taeckholmiana alcoholic root extract and cisplatin standard drug against HepG2 cells and 60 cells using the MTT assay (n = 3). Data were expressed as the mean value of cell viability (% of control) ± SD; *significantly different from the control group at P < 0.05.

Conclusions

Intriguingly, there are no reports in the available literature about the rare endangered E. taeckholmiana growing wildly in Egypt. So, preservation of this species is a must and we found that MS media supplied with 1 mg/L 2,4D and 0.5 mg/L kinetin were the best for callus formation from induced seedling explants. Moreover, different chromatographic techniques were applied for chemical profiling, where column chromatography of the defatted alcoholic root extract resulted in the isolation and identification of four metabolites. Furthermore, GC-MS of the n-hexane fraction afforded the identification of 29 components. Besides, UPLC-ESI-MS profiling of the alcoholic extract of the root and in vitro induced callus revealed that 53 compounds were tentatively identified in the root and 30 of them could be detected in the callus too. However, 15 metabolites were detected in the callus. Finally, the biological study exhibited that the root extract possessed inhibitory activity against α-amylase and α-glucosidase enzymes and moderate cytotoxic activity against HepG2.

Materials and Methods

Plant Material

E. taeckholmiana whole fruiting plant was gathered in August 2015 from the Burg El-Arab region (Alexandria Province, Egypt). Prof. Dr. A. A. Fayed, Professor of plant taxonomy, Faculty of Science, Assiut University, Egypt kindly identified the plant. A voucher specimen was kept in the Herbarium of the Faculty of Science, Cairo University, Egypt.

Tissue Culture of Seeds of E. taeckholmiana

Seed Sterilization and In Vitro Seed Germination

Surface sterilization was performed by washing the seeds placed in a cylinder with running tap water for a minute. Then, they were immersed first in 70% ethyl alcohol (Adwic, ARE) for 2 min and then in a 5% sodium hypochlorite solution for 5 min with continuous shaking. Finally, the seeds were rinsed with double-distilled sterile water three times under sterile conditions. Sterilized seeds were transferred aseptically to half-strength Murashige and Skoog (MS) media (Duchefa, Germany) (2.2 g/L) supplemented with 30 g/L sucrose (Adwic, ARE) and 6 g/L agar (Bioworld). They were incubated at 20 °C in a growth cabinet for a 16 h photoperiod. The percentage of seed germination was calculated after 20 days of cultivation using the following formula: (number of germinated seeds/total number of cultured seeds) × 100.[54]

Callus Induction

Thirty days old seedlings were aseptically cut into 1 cm pieces and transferred to MS media (4.4 g/L) with 30 g/L sucrose and 8 g/L agar, supplemented with the following plant growth regulators: medium I (1 mg/L 2,4D and 0.5 mg/L kinetin); medium II (1 mg/L NAA and 0.1 mg/L BAP); and medium III (1 mg/L NAA and 0.5 TDZ) (Sigma Chemical Co). The pH was adjusted to 5.8, and the cultures were incubated at 20 °C under a white fluorescent lamp (16 h photoperiod) for 4 weeks. The callus induction percentage was calculated using the following formula: (no. of explant-produced callus/total no. of explants cultured) × 100.[55] Four weeks old calli were subjected to subculturing using the same media composition, and they were incubated under the same conditions for 3 weeks. Growth curves were obtained by cutting the produced calli for each media separately to 2 g pieces and transferring them to a fresh medium of the same composition. The fresh weight was measured every 7 days for 4 weeks. Three replicates were made for each treatment and the mean values of three readings were plotted against time. Additionally, growth dynamics in callus cultures was calculated to evaluate the growth of the callus for each media. Growth dynamics, including growth index (GI), specific growth rate (μ), and doubling time (dt), was calculated as follows[56]

(GI) = (Ge – Gx)/Gx, where Ge is the final callus weight and Gx is the initial weight (2 g).

(μ) = (ln x – ln xo)/t, where xo is the initial callus weight (2 g) and x is the callus weight at time t (21 days).

(dt) = ln(2)/μ, where dt is the time required for callus to double and μ is the specific growth rate.

The calli grown on the media with the best growth parameters were subcultured every 3 weeks three times before they were collected for extraction.

Extraction

The dried powdered root (800 g) was cold macerated with 80% ethyl alcohol until complete exhaustion, where the total alcoholic extract was filtered and evaporated under reduced pressure at 50 °C using a Buchi rotary evaporator to afford 15 g, which was defatted to yield 2 and 11.5 g of the n-hexane fraction and the defatted total extract (semisolid residue), respectively. Additionally, about 50 g of 40 days old fresh callus grown on medium I was extracted in the same manner to give 1.2 g of viscous residue.

Column Chromatography for Isolation

Ten grams of the defatted root extract was applied as a dry-mixed initial zone to silica gel 60 (0.063–0.200 mm; Merck) column chromatograph (120 cm × 5 cm, 300 g) packed with n-hexane; the polarity was increased gradually using dichloromethane and then methanol. The resulting fractions were examined by TLC (silica gel-precoated, Kieselgel 60 F254, silica 0.25 mm, Germany) using solvents (petroleum ether/dichloromethane, 2: 1, SI), (petroleum ether/dichloromethane/methanol, 15:15:1, SII), (dichloromethane/methanol, 9:1, SIII), and (ethyl acetate/methanol/water, 10:6:1, SIV). TLC was visualized under UV light and the thymol/sulfuric acid reagent. Crystallization afforded purification of four compounds, compounds 1 (50 mg), 2 (30 mg), 3 (50 mg), and 4 (40 mg), which were subjected to melting points (Electrothermal Ltd., England), EI-MS (ISQ LT), and 1H and 13C NMR studies (Bruker, Switzerland; 100 and 400 Hz, respectively).

Gas–Liquid Chromatography-Mass Spectrometry

The n-hexane fraction of the root was analyzed by GLC-MS (Agilent 6890) with the following parameters: a fused silica capillary column: PAS-5 ms (30 m × 0.25 μm film thickness), detector: FID; temperature of the detector: 280 °C; temperature of the injector: 250 °C; recorder: dual-channel recorder; column temperature: 50–280 °C (8 °C/min); carrier gas: helium (1 mL/min); and EI: 70 eV.[57] Identification of the compounds was based on the comparison of their EI–mass spectra with the NIST 05 (National Institute of Standards and Technology) and the available literature. The peak area was used for the calculation of relative percentages of components.

UPLC-ESI-MS/MS

Root and in vitro induced callus extracts, 40 days old callus grown on medium I (100 μg/mL), were dissolved in high-performance liquid chromatography (HPLC) solvent grade and filtered using a 0.2 μm membrane disc filter. Then, 10 μL of each of the prepared samples was individually injected into the instrument equipped with a reversed-phase C18 column (ACQUITY UPLC BEH C18 2.1 mm × 50 mm Column, 1.7 μm particle size). The mobile phase was filtered using a 0.2 μm filter membrane disc and degassed by sonication before injection. The flow rate was adjusted to 0.2 mL/min using a gradient mobile phase (methanol and water acidified with 0.1% formic acid that was applied from 10 to 30% in 5 min, from 30 to 70% in 10 min, from 70 to 90% in 5 min, then the gradient was held for 2 min, and finally from 90 to 10% in 2 min). The samples were injected automatically using a Waters ACQUITY FTN autosampler. The instrument was controlled by Masslynx 4.1 software. The parameters for analysis were measured using negative modes as follows: source temperature, 150 °C; cone voltage, 30 eV; capillary voltage, 3 kV; desolvation temperature, 450 °C; cone gas flow, 50 L/h; and desolvation gas flow, 900 L/h. Mass spectra were detected in the ESI negative ion and positive ion modes between m/z 50 and 1000.

In Vitro α-Amylase Inhibitory Activity

The α-amylase enzyme solution was prepared at a concentration of 0.5 mg/mL in phosphate-buffered saline (20 mM at pH 6.9). One milliliter from various extract concentrations (7.8–1000 μg/mL) was mixed with 1 mL of the prepared enzyme and incubated at 37 °C. After 10 min, 1 mL of a 0.5% starch solution was added and incubated for another 10 min. The reaction was ended by the addition of 2 mL of dinitro salicylic acid (coloring agent) and the mixture was then heated for 5 min in a boiling water bath. The absorbance of the color produced was measured after cooling at 565 nm.[58] The results were expressed as % of inhibition and as IC50. The percent of inhibition was calculated according to the following formula

% of inhibitory activity = (1 – As/Ac) × 100, where As is the absorbance in the presence of the extract and Ac is the absorbance of control. IC50 is the concentration of the extract that inhibits 50% of enzyme activity.[58]

In Vitro α-Glucosidase Inhibitory Activity

The α-glucosidase enzyme solution was prepared at a concentration of 0.5 mg/mL in phosphate-buffered saline (20 mM at pH 6.9). One milliliter from various extract concentrations (7.8–1000 μg/mL) was mixed with 1 mL of the prepared enzyme solution and incubated at 37 °C for 10 min. After that, 20 μL of the 4-nitrophenyl-β-d-glucopyranoside substrate (P-NPG) at a concentration of 5 mM was added and incubated at 37 °C for 20 min. The reaction was ended by the addition of 50 μL of 0.1 M Na2CO3. The absorbance was measured at 405 nm. The results were expressed as % of inhibition and as IC50. The percent of inhibition was calculated as previously described.

Acarbose was used as a standard antidiabetic drug during the in vitro assay at various concentrations (7.8–1000 μg/mL). The previous measurements were carried out in triplicate and the results were expressed as mean ± standard deviation.

Human hepatocellular carcinoma (HepG2) and mouse myelogenous leukemia (M-NFS-60) cell lines, obtained from VACSERA Tissue Culture Unit, were used to evaluate the cytotoxic effect of the defatted alcoholic root extract at different concentrations (4–500 μg/mL) using the cell viability MTT assay[59] (Figures and 8). Percentages of relative viability as well as 50% inhibitory concentrations (IC50) were calculated for each cell line.

Statistical Analysis

Linear regression was performed for the calculation of IC50 in the in vitro assay. The Microsoft Excel 2010 program was used for data analysis and figure drawings. Data were performed in triplicate and presented as mean ± SD. The cytotoxicity results were expressed as the mean value of cell viability (% of control) ± SD at P < 0.05.